We initially tested whether 4u8c affects doxycycline induced mutant proinsulin GFP expression and the effect of the inhibitor on cell survival, apoptosis and XBP1 splicing. The compound kinase inhibitor Belinostat 4u8c had no effect on mutant insulin ex pression induced by doxycycline or cell viability up to 10 uM. However, at concentrations 25 uM cell loss was observed and apoptotic cells were detected as monitored by cleaved caspase 3 protein expression. Consequently, for all subsequent experiments 5 uM 4u8c was used. At this concentration XBP1 splicing in response to mutant proinsulin expression or thapsi gargin treatment was completely prevented. As expected, the inhi bitor had no effect on mutant proinsulin or thapsigargin induced activation of the PERK pathway as monitored by Ser51 phosphorylation of eIF2.

Inhibitors,Modulators,Libraries To examine the effect of IRE1 inhibition on global mRNA expression in response to misfolded proinsulin expression, we treated cells with Dox for 48 h to induce mutant proinsulin in either the presence Inhibitors,Modulators,Libraries or absence of 4u8c and performed microarray analysis from two independent experiments. The inhibitor alone did not affect gene expression changes 1. 5 fold. Doxycycline treatment lead to 1. 5 fold induction of 120 genes, most of which were previously observed to be in creased by mutant proinsulin expression. This is summarized in Figure 2 and highlighted in the top and right boxes. Surprisingly, a large subset of these genes are no longer upregulated 1. 5 fold, while 30% are still upregulated when the inhibi tor was added in the presence of Dox. However, genes that were still induced 1.

5 fold in the presence of the inhibitor usually exhibit lower expres sion than with Dox alone. The induction of only 6 genes appeared to be not af fected by the inhibitor. Thus, it appears that the IRE1 pathway contributes to the induction or maximal induc tion of the majority of genes in response to mutant pro insulin expression. Treatment Inhibitors,Modulators,Libraries with Dox for 48 h also leads to the down regulation of a number of genes 1. 5 fold. The down regulation of most of these genes is dependent on IRE1 as the presence of the inhibitor re duces or prevents the down regulation of the majority of these genes. The microarray analysis suggests that maximal induc tion of most genes is dependent on IRE1 activity. We validated some of the well Inhibitors,Modulators,Libraries established UPR genes by quantitative PCR.

As shown in Figure 3, IRE1 inhibition had no effect on the induction of the major UPR gene GRP78, but did prevent maximal induction of most of the genes examined, including SDF2L1, DNAJB9 ERdj4, HERP and EDEM1. We also examined pro apototic genes in response to Dox with or without inhibitor. Inhibitors,Modulators,Libraries CHOP mRNA levels are not significantly affected by 48 h mutant proinsulin. However, other pro apoptotic genes such as Trib3 and TxNIP that often are induced by mu tant proinsulin expression are reduced by the inhibitor.